Abstract

Breast cancer is the second largest cause of cancer-related deaths in women all over the world. Epidemiological studies suggest that the consumption of high-fat diets can promote the incidence of breast cancers in both developed and developing countries. In particular, the lipid-rich diet contains arachidonic acid (AA, a C20:4 polyunsaturated fatty acid), which has been shown to be associated with tumor formation in breast tissues. Nevertheless, the actual mechanism by which AA induces the metastatic transformation and malignancy is not well understood. The goal of my dissertation, therefore, is to identify the molecules and unravel the pathways that participate in AA-induced carcinogenesis. In this investigation, two different breast cancer cells were used—MDA-MB-231 and MCF7. Although both cells were derived from the cancerous tissues of the human breast, they display different phenotypes. For example, the estrogen-receptor-negative MDA-MB-231 cells are highly invasive, with typical fibroblast structures, whereas MCF7 cells are estrogen receptor positive and are weakly invasive, luminal epithelial cells. Briefly, both MDA-MB-231 and MCF7 cells were treated with AA (100 µM) and a lipoxygenase inhibitor (nordihydroguaiarectin acid or NDGA, 10 µM), followed by the isolation and characterization of the AA metabolites (i.e., eicosanoid molecules) by high-performance liquid chromatography (HPLC). Immunoblot and confocal microscopy were performed to elucidate the protein expression and intracellular targeting of 5-lipoxygenase (5-LOX), the enzyme that utilizes AA to synthesize LTB4 and other leukotrienes (LTs). Metastatic migration was analyzed by wound-healing and matrigel-mediated invasion assays. The involvement of lipid rafts (LRs) in inducing migration/invasion of MDA-MB-231 cells was evaluated by treating the cells with an LR disruptor, methyl-β-cyclodextrin (MBCD). The analysis of LR components (i.e., caveolin-1, flotilin and cholesterol) was also monitored. ^ The results suggest that while MDA-MB-231 cells produced high levels of prostaglandins (i.e., PGE2 and PGD2), MCF7 cells synthesized excess hydroxyeicosatetraenoic acid (HETEs) compounds. Interestingly, MDA-MB-231 cells when stimulated with AA showed increased synthesis of LTB4 (∼4-fold) and decreased the production of PGE 2 and PGD2 (∼2-fold). In contrast, AA suppressed the synthesis of PGE2, PGD2, LTB4, and HETE compounds in MCF7 cells, suggesting that AA affects the eicosanoid synthesis in MDA-MB-231 and MCF7 cells differently. Furthermore, I found that the AA treatment stimulated the expression of 5-LOX and promoted the migration and invasion by MDA-MB-231 cells. Interestingly, NDGA significantly blocked the AA-induced 5-LOX activation and cellular migration/invasion. AA also increased the phosphorylation of NFκB and produced excess IL-6 and IL-8. Again, AA showed minimal or no effects on 5-LOX, cytokines, and NFκB activation in MCF7 cells, which suggests that AA activates the different sets of molecules/pathways in non-invasive and invasive breast cancer cells, respectively. Because AA stimulated the synthesis of LTB4 in MDA-MB-231 cells, I propose that the autocrine and paracrine signaling caused by LTB4 activates NFκB-mediated cytokine release, which promotes cellular inflammation and malignancy in invasive breast cancers. The results with MBCD that blocked the invasion and migration of MDA-MB-231 cells, along with the increased synthesis of caveolin-1 and flotilin under the influence of AA, further supports the idea that the involvement of LRs is essential in AA-mediated migration and invasion of MDA-MB-231 breast cancer cells. ^ Finally, my results point out that the LTB4, which is synthesized by AA, activates the inflammatory pathways that are critical for causing the metastatic invasion of breast cancer cells. In my opinion, this is a novel finding in the field of breast cancer, and it is likely that 5-LOX pathway could be an ideal target for developing new chemotherapies to treat the patients with metastatic breast cancers.^